nanomaterials
Article
Deviation of Trypsin Activity Using Peptide
Conformational Imprints
Kiran Reddy Kanubaddi 1,† , Pei-Yu Huang 2,† , Ya-Lin Chang 2 , Cheng Hsin Wu 2 , Wei Li 2 ,
Ranjith Kumar Kankala 1,3 , Dar-Fu Tai 2, * and Chia-Hung Lee 1, *






Citation: Kanubaddi, K.R.; Huang,
P.-Y.; Chang, Y.-L.; Wu, C.H.; Li, W.;
Kankala, R.K.; Tai, D.-F.; Lee, C.-H.
Deviation of Trypsin Activity Using
Peptide Conformational Imprints.
Nanomaterials 2021, 11, 334. https://
doi.org/10.3390/nano11020334
Academic Editor: Paolo Arosio
Received: 3 December 2020
Accepted: 22 January 2021
Published: 27 January 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Nanomaterials 2021, 11, 334. https://doi.org/10.3390/nano11020334
1 Department of Life Science, National Dong Hwa University, Hualien 97401, Taiwan;
810513107@gms.ndhu.edu.tw (K.R.K.); ranjithkankala@hqu.edu.cn (R.K.K.)
2 Department of Chemistry, National Dong Hwa University, Hualien 97401, Taiwan;
610012029@gms.ndhu.edu.tw (P.-Y.H.); m9812003@ems.ndhu.edu.tw (Y.-L.C.);
m9812022@gms.ndhu.edu.tw (C.H.W.); ga025361@yahoo.com.tw (W.L.)
3 College of Chemical Engineering, Huaqiao University, Xiamen 361021, China
* Correspondence: dftai@gms.ndhu.edu.tw (D.-F.T.); chlee016@gms.ndhu.edu.tw (C.-H.L.)
† These authors contributed equally to this work.
Abstract: In this study, a methodology utilizing peptide conformational imprints (PCIs) as a tool to
specifically immobilize porcine pancreatic alpha-trypsin (PPT) at a targeted position is demonstrated.
Owing to the fabrication of segment-mediated PCIs on the magnetic particles (PCIMPs), elegant
cavities complementary to the PPT structure are constructed. Based on the sequence on targeted
PPT, the individual region of the enzyme is trapped with different template-derived PCIMPs to
show certain types of inhibition. Upon hydrolysis, N-benzoyl-L-arginine ethyl ester (BAEE) is
employed to assess the hydrolytic activity of PCIMPs bound to the trypsin using high-performance
liquid chromatography (HPLC) analysis. Further, the kinetic data of four different PCIMPs are
compared. As a result, the PCIMPs presented non-competitive inhibition toward trypsin, according
to the Lineweaver-Burk plot. Further, the kinetic analysis confirmed that the best parameters of
PPT/PCIMPs 233–245+G were Vmax = 1.47 × 10−3 mM s−1 , Km = 0.42 mM, kcat = 1.16 s−1 , and
kcat /Km = 2.79 mM−1 s−1 . As PPT is bound tightly to the correct position, its catalytic activities
could be sustained. Additionally, our findings stated that the immobilized PPT could maintain stable
activity even after four successive cycles.
Keywords: porcine pancreatic trypsin; molecularly-imprinted polymers; magnetic particles; confor-
mational imprint; secondary structure
1. Introduction
Porcine pancreatic alpha-trypsin (PPT), a proteolytic enzyme, is a pancreatic serine
protease (EC 3.4.21.4) with specificity for arginine or lysine substrate towards catalytic
hydrolysis on esters and amides, under mild reaction conditions [1–3]. Owing to these facts,
this proteolytic enzyme is often utilized in industrial and biomedical applications [1,4,5].
However, such enzymes are often immobilized into various substrates to improve stability
and reusability without affecting their activity [2,6,7]. In this vein, various enzyme immo-
bilization methods have been reported, such as covalent linkage, non-covalent adsorption,
and encapsulation systems, among others [8–12]. Nevertheless, the quest for optimum
performance is still on due to their conformational changes during immobilization [11,13].
Although an enzyme possesses a uniform structure, it often changes the conformation con-
tinuously. Consequently, the immobilized biocatalyst is organized randomly during/after
immobilization, resulting in different constitutions and a less dynamic form.
In recent times, the utilization of molecularly imprinted polymers (MIPs) has become
an emerging carrier-bound system for the immobilization of biomolecules, as they offer
reversible orientation [14]. In this context, the incorporation of MIPs with magnetic particles
https://www.mdpi.com/journal/nanomaterials
 In recent times, the utilization of molecularly imprinted polymers (MIPs) has becom
an emerging carrier-bound system for the immobilization of biomolecules, as they off
reversible orientation [14]. In this context, the incorporation of MIPs with magnetic par
cles (MPs) was first demonstrated by Ansell and Mosbach [15]. Since then, several effor
Nanomaterials 2021, 11, 334 2 of 13
have been dedicated to the utilization of MIPs for diverse applications. In a case, Ton
and colleagues applied MPs to recognize Ribonuclease A [16]. In another case, Jing an
coworkers used lysozyme as a template to adsorb blood specimens with a detection lim
(MPs) was first demonstrated by Ansell and Mosbach [15]. Since then, several efforts
at have
5 ng/mL
been [17]. Previously,
dedicated trypsin was
to the utilization of MIPsutilized as theapplications.
for diverse template toIngenerate MIPs for d
a case, Tong
assays and inhibition studies [18] .
and colleagues applied MPs to recognize Ribonuclease A [16]. In another case, Jing anda templa
ferent Nonetheless, the employment of such
forcoworkers
MPs wasused restricted
lysozyme toasthe usage of
a template to the whole
adsorb bloodprotein.
specimens with a detection limit at
5 ng/mL
Considering these facts, herein, we demonstrate to
[17]. Previously, trypsin was utilized as the template angenerate
elegantMIPs for different
method to immobili
assays and inhibition studies [18]. Nonetheless, the employment of such a template for
PPT. As segment-mediated MIPs were fabricated on MPs, cavities complementary to PP
MPs was restricted to the usage of the whole protein.
structure were constructed on those nanomaterials. As the imprinting of a random co
Considering these facts, herein, we demonstrate an elegant method to immobilize
peptide
PPT. As successfully generates
segment-mediated MIPsthewere
desired nano-cavity
fabricated on MPs,for the corresponding
cavities complementarypeptide/prto
tein [19,20], recently, a helical peptide was utilized as
PPT structure were constructed on those nanomaterials. As the imprintinga template to generate
of a random helical cav
coil
ties peptide
with highsuccessfully
affinity forgenerates
the targettheprotein,
desired having
nano-cavity for thesatisfactory
achieved corresponding pep- [21]. A
results
tide/protein [19,20], recently, a helical peptide was utilized as a template
cordingly, in this work, several PPT peptide segments were selected to generate hel to generate helical
cavities with high affinity for the target protein, having achieved satisfactory results [21].
cavities, using peptide conformational imprints decorated over the magnetic particl
Accordingly, in this work, several PPT peptide segments were selected to generate helix
(PCIMPs). This peptide
cavities, using study isconformational
divided intoimprints
four main decorated (i) Fabricating
steps: over the magneticpeptide
particlesconform
tional imprint
(PCIMPs). This(PCIs)
studyon MPs carriers;
is divided into four (ii) adsorbing
main steps: (i)trypsin to PCIMPs;
Fabricating (iii) analyzing t
peptide conforma-
binding kinetics
tional imprint of immobilized
(PCIs) on MPs carriers; PPT; and (iv) evaluating
(ii) adsorbing the reusability
trypsin to PCIMPs; of immobiliz
(iii) analyzing the
binding
PPT. The kinetics of immobilized
immobilization of PPTPPT; and on
based (iv)the
evaluating
PCIs was the carried
reusability
outofasimmobilized
illustrated in
PPT. The
Figure 1. immobilization of PPT based on the PCIs was carried out as illustrated in Figure 1.

1. Scheme illustrating the fabrication of peptide conformational imprints (PCIs) on magnetic particles (MPs) and
FigureFigure
1. Scheme illustrating the fabrication of peptide conformational imprints (PCIs) on magnetic particles (MPs) and
their binding to porcine pancreatic alpha-trypsin (PPT).
their binding to porcine pancreatic alpha-trypsin (PPT).
2. Materials and Methods
2. 2.1.
Materials
Reagentsand Methods
and Chemicals
2.1. Reagents and Chemicals
3-(Aminopropyl) trimethoxysilane (APTMS) and ammonium acetate (NH4 Ac) were
obtained from Acros Ltd. (Fair Lawn, New Jersey, United States). Iron (III) chloride
3-(Aminopropyl) trimethoxysilane (APTMS) and ammonium acetate (NH4Ac) we
hexahydrate (FeCl3 ·6H2 O) and triethylamine (TEA) were purchased from Merck Ltd.
obtained fromGermany).
(Darmstadt, Acros Ltd.
N,N(Fair Lawn,bisacrylamide
0 -Ethylene New Jersey,(EBAA)
Unitedand States). Ironacrylamide
N-benzyl (III) chloride hex
hydrate
(BAA) were 36H2O)
(FeClacquired andLancaster
from triethylamine (TEA)
(Lancashire, were
UK). purchased(GA)
Glutaraldehyde from Merck
was Ltd. (Darm
obtained
stadt,
fromGermany). N,N’-Ethylene
Ferax (Berlin, Germany). Allbisacrylamide
Fmoc-amino acids(EBAA)
were and
purchasedN-benzyl
fromacrylamide
BAChem (BA
(Bubendorf, Switzerland). Acrylamide (AM), acetic acid, N-benzoyl-L-arginine
were acquired from Lancaster (Lancashire, UK). Glutaraldehyde (GA) was obtained fro ethyl
ester(Berlin,
(BAEE), Germany).
sodium cyanoborohydride (NaBH N,N,N 0 ,N 0 -tetramethyl ethylene
Ferax All Fmoc-amino 3 CN),
acids were purchased from BAChem (Bube
diamine (TEMED), tris (hydroxymethyl) amino-methane, porcine pancreatic trypsin (PPT),
dorf, Switzerland). Acrylamide (AM), acetic acid, N-benzoyl-L-arginine ethyl est
Tween® 20, and urea were acquired from Sigma Co. Ltd. (St. Louis, MO, USA). Acetone,
(BAEE), sodium
acetonitrile cyanoborohydride
(ACN), (NaBH
dichloromethane (DCM), 3CN), N,N,N’,N’-tetramethyl ethylene diami
N,N-dimethyl formamide (DMF), piperidine,
(TEMED), tris (hydroxymethyl) amino-methane, porcine pancreatic trypsin (PPT
Nanomaterials 2021, 11, 334 3 of 13

and toluene of High-Performance Liquid Chromatography (HPLC) grade were used.

Purified distilled water acquired from a Milli-Q water purification system was used in all
the experiments.

2.2. Template Synthesis

The peptide segments, such as PPT107–116 (KLSSPATLNS), PPT145–155 (KSSGSSYPSLL),
PPT169–178 (KSSYPGQITG), and PPT233–245+G (NYVNWIQQTIAANG), were produced
through the Fmoc (fluorenylmethoxycarbonyl) solid-phase peptide synthesis approach
using a Discover SPPS Microwave Peptide synthesizer (Kohan Co. Ltd., Taipei, Taiwan)
available at the National Dong Hwa University (Hualien, Taiwan) [22].

2.3. Preparation of PCIs on MPs

2.3.1. Construction of Fe3 O4 @APTMS-GA
The synthesis of the Fe3 O4 precursor, and subsequent immobilization of amine func-
tionality, Fe3 O4 @APTMS, were performed as described previously [23,24]. Further, glu-
taraldehyde (GA) was coupled with Fe3 O4 @APTMS to construct stable secondary amine
nanoparticles. Briefly, 100 mg of Fe3 O4 @APTMS was initially placed in 50 mL of ACN
and subjected to ultrasonication for 30 min. Then, 162 µL of GA was added to the mixture.
Further, a few drops of acetic acid were added to maintain the weakly acidic state of the
reaction mixture, and stirring was performed for 2 h. Subsequently, 200 mg of NaBH3 CN
were added, and vigorous stirring was executed for another 2 h to make the reaction mix-
ture weakly alkaline. Finally, the resultant particles were recovered with a strong magnet,
washed several times with a solvent mixture of (H2 O:ACN = 1:1), and dried under vacuum.

2.3.2. Synthesis of Fe3 O4 @APTMS-GA-Acrylate

To prepare Fe3 O4 @APTMS-GA-acrylate, 300 mg of GA-modified MPs were initially
dispersed in 25 mL of dry DCM and stirred for 15 min after adding TEA (0.48 mL). Then,
acryloyl chloride (0.3 mL, 3.75 mmol) was added in a drop-wise manner to the mixture at
0 ◦ C under N2 purge and stirred for 24 h. Finally, the resultant product was washed with
DCM and dried under vacuum.

2.3.3. Preparation of PCIMPs

To prepare PCIMPs, initially, 211.2 mg of N, N’-ethylene bisacrylamide (EBAA),
56.4 mg of benzyl acrylamide (BAA), and 25.2 mg of acrylamide (AA) were dissolved
in a solvent mixture containing 16 mL of PBS (pH-7.6, 20 mM) and 2 mL of ethanol. Then,
7.5 µmol of template molecules (PPT107–116 , PPT145–155 , PPT169–178 , and PPT233–245+G ) were
dissolved separately in 20 mL of a solvent mixture of TFE and PBS at a ratio of 7:3 to
exhibit the helical structure in the polymerization system. Further, the above two reaction
mixtures were mixed after a while, and 90 mg of Fe3 O4 @APTMS-GA-Acrylate was added
to make a pre-self-assembly reaction mixture. Then, 240 µL (10%, w/w) of ammonium
persulfate and 90 µL (5%, w/v) of TEMED were added to the reaction and stirred for
24 h in the presence of N2 at RT. The template removal was performed based on previous
studies [25,26]. According to the following articles, acetic acid as a solvent disrupts the
electrostatic interactions between the template and the polymer matrix, which can be sepa-
rated. Notably, the template removal process could be achieved in few minutes. Finally, the
polymer-MPs were obtained and washed with 25 mM urea (aq) containing 5% acetic acid
and 0.5% tween-20 to remove the template. Subsequently, the pore structures formed after
the removal of the four different templates were denoted as PCIMPs107–116 , PCIMPs145–155 ,
PCIMPs169–178 , and PCIMPs233–245+G , respectively.
Nanomaterials 2021, 11, 334 4 of 13

2.4. Determination of Binding Affinities of PCIMPs

Notably, the binding experiments were carried out in 10 min to avoid adsorption at
non-specific binding sites on PCIMPs. Briefly, 10 mg of PCIMPs was added to PBS (pH-7.6,
20 mM) containing PPT at different concentrations (0.125, 0.25, 0.5, 1, and 1.5 mg/mL) and
the resulting mixture was shaken for 10 min. Then, 200 µL of supernatant was collected
and measured by Fluorescence Microplate Reader at λex/λem = 290 nm/350 nm. Each
experiment was repeated three times, and the results of the binding studies were evaluated
using the Scatchard Equation (1) [27–29].

[RL]/[L] = (Bmax − [RL])/Kd (1)

where [L] is the concentration of PPT in the solution, [RL] is the concentration of bound PPT,
Bmax denotes the maximum number of binding sites, and Kd is the dissociation constant of
the ligand.

2.5. Activity Assay of PPT and Immobilized PPT (PPT/PCIMPs)

The catalytic activity of PPT and PPT/PCIMPs was measured using the HPLC method.
N-benzoyl-L-arginine ethyl ester (BAEE) was utilized as the starting material, while the
product was N-Benzoyl-L-Arginine (BA), which was observed with time. The percentage
of hydrolysis rate was calculated using the following Equation (2):

Product area ratio

Hydrolysis rate (%) = × 100 (2)
(Starting area ratio + product area ratio)

For determining the catalytic activity of PPT, initially, 1 mL mixtures possessing

different BAEE concentrations (0.5, 1.0, and 1.5 mM) were prepared using 50 mM of Tris-
HCl buffer, with a pH equal to 7.6. Then, 20 µL of 1mM HCl containing 30 µg PPT was
formulated. The assay was started by adding 20 µL of 1 mM HCl/30 µg PPT to 1 mL
mixtures with the three BAEE concentrations mentioned above, respectively. For every min,
40 µL of the solution was collected from the reaction mixture and dissolved in 500 µL of
ACN: buffer = 15:85, and 99.5 µL of the resultant solution was injected for HPLC detection
until the end of the reaction.

2.6. PPT/PCIMPs Activity Assay

Briefly, 10 mg of each PPT/PCIMPs were separately added to 8.8 mL of the BAEE
solutions (0.5, 1.0, and 1.5 mM), and for every min, 80 µL of that solution were separated
from the mixture and dissolved in 1 mL of ACN: buffer = 15:85. From this, 99.5 µL of
the solution was collected for HPLC detection until the end of the reaction. The same
procedure was also carried out for the reusability test.
An intelligent, high-performance liquid chromatography (HPLC, model L7100, Hi-
tachi, Tokyo, Japan) set-up equipped with a UV detector (Hitachi model L-2420, Tokyo,
Japan), an autosampler (Hitachi L-2200, Tokyo, Japan), and a Vercopak-RP C18 column
(Vercotech Corp., Taipei, Taiwan) was used to determine the purity of peptides and for per-
forming the kinetic analysis of the immobilized enzyme. In the hydrolysis test, the mobile
phase of HPLC was composed of 0.38 mL of phosphoric acid, 0.47 mL of triethylamine,
and 1 L of DI-H2 O. The solution was then adjusted to pH 2.4 with NaOH and HCl. The
ultraviolet wavelength was set at 214 nm.

2.7. Determination of Kinetic Constants of PPT and PPT/PCIMPs

The kinetic parameters of PPT and PPT/PCIMPs were evaluated from the Michaelis–
Menten plot obtained from the following Equation (3),

Vmax [S]
ν= (3)
(Km + [S])
 The turnover number (kcat) was calculated using the below Equati
Nanomaterials 2021, 11, 334 5 of 13
𝑘 = 𝑉 /[𝐸 ]
where[E] is the enzyme concentration [13].
where v is the reaction velocity at [S], V max is the maximum rate of the reaction, Km is the
Michaelis half-saturation constant, and [S] is the concentration of the substrate.
3. Results andnumber
The turnover Discussions
(kcat ) was calculated using the below Equation (4).

3.1. Rational Selection of the kTemplate

cat = Vmax / [ E ] (4)

whereThe template
[E] is the for the imprinting
enzyme concentration [13]. was chosen considering the foll
(i) Peptide segments from the flank part of the PPT spatial structure w
3. Results and Discussions
template. Due toof the
3.1. Rational Selection five disulfide linkages connected among PPT, the cho
Template
ments Theable
template to for
influence catalysis
the imprinting is limited.
was chosen considering(ii)
theThe length
following of the pepti
parameters:
(i) Peptide segments from the flank part of the PPT spatial structure were selected as
template is a significant parameter. For instance, short peptide resi
the template. Due to five disulfide linkages connected among PPT, the choice of peptide
structures
segments ablethat can help
to influence the isimprinting
catalysis and
limited. (ii) The protein-rebinding
length of the peptide segments processe
in the template is a significant parameter.
in this study, four PPT peptides, specifically PPT For instance, short peptide
107–116 residues
, PPT form
145–155 , PPT1
flexible structures that can help the imprinting and protein-rebinding processes [19,30].
were chosen.
Therefore Thefour
in this study, locations of these
PPT peptides, segments
specifically PPT107–116are
, PPTshown in169–178
145–155 , PPT Figure
, 2.
233–245 , were chosen. The locations of these segments are shown in Figure 2. At one
PPT
and PPT
233–245 peptide, a glycine (G) residue was added to make a stable
end of the PPT233–245 peptide, a glycine (G) residue was added to make a stable peptide
flexibility [31,32].
chain with flexibility [31,32].

Figure 2. The structure of porcine pancreatic trypsin (cylinder: α-helix; arrow: β-sheet). The selected
Figure
sequences2.areThe structure
in yellow. of porcine
These segments consistpancreatic trypsin
of the series: i.e., (cylinder:
PPT107–116 , PPT145–155 , α-helix;
PPT169–178 , arrow:
lected
and PPTsequences arestructure
233–245 . The crystal of PPTThese
in yellow. segments
was reproduced fromconsist of the series: i.e., PPT10
http://www.ncbi.nlm.nih.gov/
and PDB
PPT ID:, 1S81
169–178 and[33].
PPT233–245. The crystal structure of PPT was reproduced from
http://www.ncbi.nlm.nih.gov/
3.2. Analysis of the Template and PDB ID: 1S81 [33].
Furthermore, the template was synthesized using a CEM Discover Microwave Syn-
thesizer (Kohan Co., Taipei, Taiwan) at National Dong Hwa University (Hualien, Taiwan).
3.2. Analysis of the Template
The peptide segments PPT107–116 , PPT145–155 , PPT169–178 , and PPT233–245+G were selected as
Furthermore,
templates. Initially, these the template
segments was synthesized
were fabricated using the Fmocusing a CEM
solid-phase peptideDiscov
synthesis [22]. Further, the purity of the template molecules was confirmed by HPLC
thesizer (Kohan Co., Taipei, Taiwan) at National Dong Hwa Univer
wan). The peptide segments PPT107–116, PPT145–155, PPT169–178, and PPT233
as templates. Initially, these segments were fabricated using the Fmoc s
 Nanomaterials 2021, 11, 334 6 of 13

equipped with an RP-18 (flow rate- 1 mL/min). Among the selected peptide segments,
PPT107–116 and PPT169–178 showed a purity higher than 96%. Contrarily, the other two seg- 6 of 13
Nanomaterials 2021, 11, x FOR PEER REVIEW
ments, PPT145–155 and PPT233–245+G , had a lower purity of around 88%, which could be
attributed to their longer length, leading to a difficulty in the purification of those peptide
segments [34]. Further, the molecular mass of the template was analyzed using a Matrix-
Assisted
(DHB). TheLaser Desorption/Ionization-Time
reported m/z values of PPT107–116 of, Flight (MALDI/TOF)
PPT145–155, PPT169–178, andmassPPT
spectrometer
233–245+G were ob-

(MS) (Bruker,
served Bremen,
at 1017.69, Germany)
1125.56, 1037.39,with a matrix
and 1614.24 consisting
[M + Na] of+,2,5-dihydroxybenzoic
respectively. Further, acid
the pep-
(DHB). The reported m/z values of PPT 107–116 , PPT145–155 , PPT169–178 , and PPT233–245+G
tide segment PPT 233–245+G was analyzed with a (JASCO, J-715, Tokyo, Japan) circular di-
were observed at 1017.69, 1125.56, 1037.39, and 1614.24 [M + Na]+ , respectively. Further, the
chroism (CD) spectrometer to validate the helix structure in the mixtures of buffer and
peptide segment PPT233–245+G was analyzed with a (JASCO, J-715, Tokyo, Japan) circular
TFE (Figure 3). Usually, the helical peptides possess negative bands at 208 and 225 nm in
dichroism (CD) spectrometer to validate the helix structure in the mixtures of buffer and
a TFE
mixture of 3).
(Figure PBS and TFE,
Usually, while peptides
the helical the peptides
possesswith random
negative bandscoilatstructures
208 and 225show
nm innegative
a
bands
mixture of PBS and TFE, while the peptides with random coil structures show negative were
at 200 nm in PBS [35]. After that analysis, the selected PPT peptide segments
used
bandsto at
generate
200 nm helix
in PBScavities using
[35]. After thatthe PCIMPs-based
analysis, the selectedapproach.
PPT peptide segments were
used to generate helix cavities using the PCIMPs-based approach.

10

PBS:TFE = 7:3
PBS:TFE = 3:7
Ellipticity (mdeg)

5
PBS

-5

180 200 220 240 260 280

Wavelength (nm)
Figure 3. The circular dichroism (CD) spectrum of the PPT 233–245+G segment in different
Figure
solvent3.systems.
The circular dichroism (CD) spectrum of the PPT 233–245+G segment in different solvent
systems.
3.3. Characterization of MPs and PCIMPs
3.3.1.
3.3. FTIR Analysisof MPs and PCIMPs
Characterization
A Fourier-transform
3.3.1. FTIR Analysis infrared spectrometer (FTIR, Bruker TENSOR 27, Ettlingen, Ger-
many) was employed to examine the successive surface modifications on MPs (Figure 4).
TheA Fourier-transform
peaks at 586 cm−1 and infrared
3444 cm−spectrometer
1 can be ascribed (FTIR,
to Fe-OBruker TENSOR
stretching 27, Ettlingen,
vibration and O-H Ger-
many) was of
stretching employed to examine
Fe3 O4 (Figure 4a). Thethe successivepeaks
characteristic surface modifications
of silanol on MPs
groups (Si-O-H) on(Figure
the 4).
The peaks
surface of at
Fe3586
O4 atcm1030
−1 and − 1
cm3444 , ascm −1 canatbe
well as −
ascribed
1100 1
cm , and to Fe-O stretching
the peak − 1
vibration
at 3421 cm and O-H
can rep-
resent the of
stretching characteristic
Fe3O4 (Figure peaks4a).
of NH
The2 (primary amine)peaks
characteristic of APTMS, indicating
of silanol the successful
groups (Si-O-H) on the
modification
surface of Fe3Oof4 at
the1030
Fe3 Ocm4 nanoparticles
−1, as well as surface
at 1100with
cm−1amine
, and thegroups
peak(Figure
at 34214b)
cm [36–38].
−1 can repre-
Additionally, the peaks near of 3413 cm −1 can represent the existence of the N-H func-
sent the characteristic peaks of NH2 (primary amine) of APTMS, indicating the successful
tional group, and no peak at 1739 cm−1 can indicate the C=O group at both ends of the
modification of the Fe3O4 nanoparticles surface with amine groups (Figure 4b) [36–38].
glutaraldehyde molecule reacted with NH−12 , attributed to the established stable secondary
Additionally, the peaks near of 3413 cm can represent the existence of the N-H functional
structure. The secondary amine-modified iron nanoparticles are more reactive than the
group,
primaryand no peak
amine at 1739
because cm−1 caneffect
the inductive indicate the C=O amine
of secondary groupmakes
at both ends
them of the
more glutaral-
stable
dehyde molecule reacted with NH
compared to the primary amine (Figure 4c). The peak at 1619 cm can be ascribed to the struc-
2, attributed to the established
− 1 stable secondary
ture. The secondary
characteristic peak of amine-modified iron nanoparticles
C=C, indicating successful acrylation ofare more(Figure
the MPs reactive
4d) than
[39]. the pri-
mary amine because the inductive effect of secondary amine makes them more stable
compared to the primary amine (Figure 4c). The peak at 1619 cm−1 can be ascribed to the
characteristic peak of C=C, indicating successful acrylation of the MPs (Figure 4d) [39].
Nanomaterials 2021, 11, x FOR PEER REVIEW 7 of 13
Nanomaterials
Nanomaterials 2021,
2021, 11,
11, x334
FOR PEER REVIEW 77of
of13
13

55
55 (d)
(d)
50
50
45
45 _1
C=C (1619
_1cm )
4040 C=C (1619 cm )

(%) Transmittance
(%) Transmittance
(c)
3535 (c)

3030
__
2525 (b)
(b) N-H (3413
N-H
11
cm ) )
(3413cm

2020
_1
15 Si-O-H (1030-1100 cm ) _1
15 (a) Si-O-H (1030-1100 cm )
(a)
10
10
5 O-H (3444 cm
_1
) Fe-O (586 cm
_1
)
5 O-H (3444 cm
_1
) Fe-O (586 cm
_1
)
4000 3500 3000 2500 2000 1500 1000 500
4000 3500 3000 2500
Wavenumber 2000
(cm_1) 1500 1000 500
Wavenumber (cm_1)
Figure 4. FTIR spectra of (a) Fe3O4, (b) Fe3O4@APTMS, (c) Fe3O4@APTMS-GA, and (d)
Figure 4. FTIR spectra of (a) Fe3 O4 , (b) Fe3 O4 @APTMS, (c) Fe3 O4 @APTMS-GA, and
Figure 4. FTIR spectra of (a) Fe3O4, (b) Fe3O4@APTMS, (c) Fe3O4@APTMS-GA, and (d)
Fe3O4@APTMS-GA-acrylate.
(d) Fe3 O4 @APTMS-GA-acrylate.
Fe3O4@APTMS-GA-acrylate.
3.3.2.
3.3.2.FE-SEM
FE-SEMAnalysis
Analysis
3.3.2. FE-SEM
The
The surface Analysis
surface morphology
morphologyof of various
various MPsMPs and
and PCIMPs
PCIMPs was was analyzed
analyzed using using aa Field
Field
Emission
Emission Scanning
The surfaceScanning Electron Microscope
Electron Microscope
morphology (FE-SEM,
of various(FE-SEM,
MPs andJEOLJEOLJSM-7000F/JEOL
PCIMPsJSM-7000F/JEOL
was analyzed Ltd., Tokyo,
Ltd.,using
Tokyo, Ja-a Field
pan) (Figure
Japan) (Figure 5). As a
5). As result, it was
a result, observed
it was observed that the fabricated
that the Fe
fabricated O particles were
Fe3 O4 particles spher-
Emission Scanning Electron Microscope (FE-SEM, JEOL 3 4
JSM-7000F/JEOL Ltd.,were Tokyo, Ja-
ical, showing
spherical, a uniform
showing size size
a uniform distribution withwith
distribution an anaverage
average size of of
size ~237
~237 nmnm(Figure
(Figure5a). 5a).
pan) (Figure 5). As a result, it was observed that the fabricated Fe3O4 particles were spher-
Further,
Further, APTMS
APTMS immobilization
immobilization on Fe33O O44nanoparticles
nanoparticlesresulted
resultedin insubstantial
substantial changes
changes in in
ical,
theshowing
the size
size and a uniform
and shape of thosesize
MPs,distribution
MPs, havingincreased
having withtheir
increased an average
their average
averagesize size
sizetotoof ~237
~278
~278 nm nm (Figure
nm(Figure 5b). 5a).
Further, APTMS
The subsequent
(Figure immobilization
immobilization
5b). The subsequent ofon Fe3O4 nanoparticles
glutaraldehyde
immobilization on the MPs
of glutaraldehyde resulted
on thein
resulted MPssubstantial
in resulted
an increase changes
in in
an in
the sizeaverage
their
increase and shape
in their toof~309
sizeaverage those
nmMPs,
size (Figure
to ~309 having
5c). increased
nm Notably,
(Figure their
a slight
5c). average
a slightsize
aggregation
Notably, can toobserved
be ~278 nm
aggregation can after
be
(Figure
observed 5b).
the successive The subsequent
after surface immobilization
modification
the successive surface onmodification of
the MPs. Thisonglutaraldehyde
could be because
the MPs. on the MPs
nanoparticles
This could resulted
be because na- in an
treated
with different
increase
noparticles in their solvents
treated withand
average drytosamples
size
different ~309 nm
solventswere collected
(Figure
and 5c).after
dry samples the
Notably,
were surface
a slight
collectedmodification.
aggregation
after the sur- Thecan be
dry modification.
face
observed power shows
after strong
the The dry aggregation,
successivepower as reported
showsmodification
surface strong in on
previous
aggregation, theasMPs.studies
reportedThisin [40]. Further,
previous
could the na-
stud-
be because
acrylate
ies [40]. monomer
Further, the conjugation
acrylate
noparticles treated with different107–116 with
monomer MPs resulted
conjugation in
solvents and 145–155an average
with MPs size of
resulted
dry samples were169–178 ~323
in nm
an (Figure
average 5d).
size
collected after the sur-
Amongst
of ~323 nmall PCIMPs,
(Figure the PCIMPs
5d). Amongst all ,PCIMPs,
PCIMPs the PCIMPs , and PCIMPs
107–116, PCIMPs have shown
145–155 , and stud-
face modification. The dry power shows233–245+G strong aggregation, as reported in previous
similar
PCIMPs size
169–178at ~370 nm, whereas PCIMPs
have shown similar size at ~370 nm, whereas PCIMPs were comparatively larger
233–245+G at ~408
were compara- nm
ies(Figure
[40]. Further,
5e–h).
the acrylate monomer conjugation with MPs resulted in an average size
tively larger at ~408 nm (Figure 5e–h).
of ~323 nm (Figure 5d). Amongst all PCIMPs, the PCIMPs , PCIMPs , and 107–116 145–155

PCIMPs169–178 have shown similar size at ~370 nm, whereas PCIMPs233–245+G were compara-
tively larger at ~408 nm (Figure 5e–h).

Figure
Figure 5.
5. FE-SEM
FE-SEMimages imagesofof(a)
(a)Fe
Fe3OO4, (b) Fe3O4@APTMS, (c) Fe3O4@APTMS-GA, (d) Fe3O4@APTMS-GA-acrylate, (e)
3 4 , (b) Fe3 O4 @APTMS, (c) Fe3 O4 @APTMS-GA, (d) Fe3 O4 @APTMS-GA-acrylate,
PCIMPs 107–116, (f) PCIMPs145–155, (g) PCIMPs169–178, and (h) PCIMPs233–245+G (Scale bar: 100 nm).
(e) PCIMPs107–116 , (f) PCIMPs145–155 , (g) PCIMPs169–178 , and (h) PCIMPs233–245+G (Scale bar: 100 nm).

Figure 5. FE-SEM images of (a) Fe3O4, (b) Fe3O4@APTMS, (c) Fe3O4@APTMS-GA, (d) Fe3O4@APTMS-GA-acrylate, (e)
PCIMPs107–116, (f) PCIMPs145–155, (g) PCIMPs169–178, and (h) PCIMPs233–245+G (Scale bar: 100 nm).
Nanomaterials 2021, 11, 334 8 of 13

3.4. Binding Studies of PCIMPs

For comparison, the binding affinities of the PPT to each PCIMPs were measured by
the linear regression curve based on the Scatchard equation. As shown in Table 1, the
PCIMPs 233–245+G had the lowest Kd value (0.21 µM) of all the PCIMPs. It was observed
from the results that the Kd values showed a decreasing trend with an increase in the
number of peptide residues. Therefore, the higher the number of peptide segments in the
template, the better the observed binding affinities. For instance, for the 14-mer peptide,
the Kd was 0.21 µM, and it showed better affinity when compared to the 10 and 11-mer
peptides [19]. Similarly, for PCIMPs145–155 , the Kd value was 0.38 µM, and it presented a
better affinity than that of a 10-mer peptide. On the other hand, both PCIMPs107–116 and
PCIMPs169–178 have shown a similar number of peptide residues in the template. In this
case, affinities of the PPT to PCIMPs were more closely related to the molecular weight of
the template residues. For example, the Kd value of the PCIMPs169–178 was 0.55 µM, which
showed a better binding affinity than PCIMPs107–116 (0.65 µM).

Table 1. Binding affinity values of various PCIs on magnetic particles (PCIMPs) to PPT.

MPs PCIMPs107–116 PCIMPs145–155 PCIMPs169–178 PCIMPs233–245+G

Residue 10 11 10 14
[Kd ] µM 0.65 0.38 0.55 0.21
[Bmax ] nM 0.75 1.11 0.95 1.11
Bmax /Kd 1.15 2.92 1.73 5.29

Previously, Griffete and colleagues developed a magnetic-protein imprinted poly-

mer (M-PIP) by combining photopolymerization with a grafting approach onto surface-
functionalized MPs. The authors demonstrated that the green fluorescent proteins were
bound to MIPs in less than 2 h with a high affinity (Kd = 0.29 µM) [41]. In another study,
MIPs were synthesized using a solid-phase approach on metal chelate functionalized glass-
beads to immobilize trypsin using its surface histidine. Although less cross-reactivity with
other proteins was observed, the dissociation constant value of the MIP-trypsin complex
was 0.237 µM [42], with a lagging binding capacity. Notably, in this study, the PCIs devel-
oped on the surface of MPs create recognition sites that are complementary to the protein
conformational structure and, therefore, significantly increase the specificity toward the
targeted protein. The best binding performance of PCIMPs233–245+G occurred in 10 min
with a high affinity (Kd = 0.21 µM). Upon a comprehensive evaluation of binding affinities
and absorption time, it was apparent that conformational imprints on MPs acquired better
results in these protein-imprinted particles. Together, our findings indicated a higher
affinity of protein (PPT) to PCIMPs 233–245+G (Kd = 0.21 µM), in comparison to the other
MIPs grafting methods.

3.5. Kinetic Parameters of PPT and PPT/PCIMPs

In addition, the PCIMPs bound to PPT exhibited excellent catalytic activity. To demon-
strate this aspects, the kinetic parameters of PPT and PPT/PCIMPs were explored by
varying the BAEE substrate concentration (0.5–1.5 mM). They were then calculated using
the Michaelis-Menten plot (Figure 6a). As shown in Table 2, among all the PPT/PCIMPs,
PPT/PCIMPs 233–245+G had the best kinetic parameters. The Km value of PPT (0.36 mM)
was almost similar to that of the PPT/PCIMPs 233–245+G (0.42 mM), which could be due to
the high feasibility of forming an enzyme-substrate complex, and also a lower diffusion
restraint imposed on the flow of the substrate and product molecules from the grafted
polymer matrix of the MPs [43,44]. The Vmax values were found to be 3.2 × 10–3 mMs−1 and
1.47 × 10–3 mMs−1 for PPT and PPT/PCIMPs 233–245+G , in which the Vmax was decreased
for PPT/PCIMPs when compared to the free enzyme. The plausible reason might be due
to the created steric hindrances that restrict the substrates’ transport, enhance diffusional
creation limitations, and decrease the enzyme’s catalytic properties. These conclusions are
in agreement with the results reported literature [45,46].
 Vmax (mM s−1) 3.2 × 10–3 0.53 × 10–3 1.25 × 10–3 0.84 × 10–3 1.47 × 10–3
[Km] mM 0.36 0.52 0.46 0.44 0.42
kcat (s )
−1 2.6 0.62 0.99 0.78 1.16
kcat/Km (mM −1 s ) 2021, 11,
−1
Nanomaterials 7.32
334 1.19 2.15 1.77 2.79
9 of 13
Note: PPT= porcine pancreatic alpha-trypsin, PPT/PCIMPs = immobilized PPT.

Figure 6.Figure 6. (a) Michaelis-Menten

(a) Michaelis-Menten andand
(b) (b) Lineweaver-Burk plots
Lineweaver-Burk plots of
ofPPT
PPTand PPT/PCIMPs
and PPT/PCIMPs obtained with with
obtained various N-benzoyl-
various N-benzoyl-
L-arginine
L-arginine ethyl (BAEE)
ethyl ester ester (BAEE) solutions
solutions (1.5,
(1.5, 1, 1,0.5
0.5mM).
mM). Note:
Note: The
Thestandard
standarddeviation for the
deviation forMichaelis-Menten
the Michaelis-Menten kinetics kinetics
plot of 1 ×1 10 –4 233–245+G is 1.41067 × 10–5 ,
plot of PPT inPPT in different
different concentration
concentration (1.5mM
(1.5 mM to to 0.5
0.5mM)
mM)is is × 10whereas and PPT/PCIMPs
–4 whereas and PPT/PCIMPs 233–245+G is 1.41067 × 10–5,
7.2111 × 10 –6 , and 5.50757 × 10 –6 for (1.5, 1, and 0.5 mM).
7.2111 × 10 , and 5.50757 × 10 for (1.5, 1, and 0.5 mM).
–6 –6

Table 2. Kinetic parameters obtained from the Michaelis-Menten plot.

Respectively, it was107–116
observed that the145–155 kcat value of PPT/PCIMPs 233–245+G was lower than
MPs PPT PPT/PCIMPs PPT/PCIMPs PPT/PCIMPs169–178 PPT/PCIMPs233–245+G
that of −PPT. The decrease of kcat values upon immobilization of enzymes are frequently
Vmax (mM s−1 ) 3.2 × 10 3 0.53 × 10−3 1.25 × 10−3 0.84 × 10−3 1.47 × 10−3
[Km ] mM reported
0.36 [13,47,48]. These
0.52 findings suggest
0.46 a limited diffusion
0.44 of the substrate
0.42 to the active
kcat (s−1 ) site and
2.6 higher structural
0.62 rigidity of the 0.99 immobilized PPT. 0.78 Our results are 1.16quite compara-
kcat /Km (mM−1 s−1 ) 7.32 1.19 2.15 1.77 2.79
ble and in agreement with the ones reported in the literature [47–50]. Furthermore, the
Note: PPT= porcine pancreatic alpha-trypsin, PPT/PCIMPs = immobilized PPT.
trypsin inhibition by PCIMPs was investigated by performing enzyme assays in the Tris-
233–245+G was lower
HCl buffer at pH 6.2,
Respectively, using
it was BAEEthat
observed as the
thekcat
substrate at various concentrations
value of PPT/PCIMPs . The Lin-
than that
eweaver of PPT.
Burk plotThe
(1/V decrease
o versus of k1/S)
cat values
is asupon
shown immobilization
in Figure 6b. of enzymes
It revealsare that
frequently
the PCIMPs
reported [13,47,48]. These findings suggest a limited diffusion of the substrate to the active
exhibited non-competitive inhibition towards trypsin, in which the PCIMPs acted as in-
site and higher structural rigidity of the immobilized PPT. Our results are quite comparable
hibitors, while the BAEE functioned as a substrate. In non-competitive inhibition, the re-
and in agreement with the ones reported in the literature [47–50]. Furthermore, the trypsin
spective inhibitors bind
inhibition by PCIMPs wastoinvestigated
the free enzyme and the
by performing enzyme-substrate
enzyme complex
assays in the Tris-HCl bufferwith the
same at affinity. Further,
pH 6.2, using BAEEthe as inhibitor
the substrate reduces the concentrations.
at various activity of the The enzyme and binds
Lineweaver Burk equally
plot (1/V versus
well to the substrate
o 1/S) is
[51,52]. as shown in Figure 6b. It reveals that the PCIMPs exhibited
non-competitive inhibition towards trypsin, in which the PCIMPs acted as inhibitors,
3.6. while the BAEE functioned as a substrate. In non-competitive inhibition, the respective
Reusability
inhibitors bind to the free enzyme and the enzyme-substrate complex with the same
Additionally,
affinity. theinhibitor
Further, the reusability
reducesofthePPT/PCIMPs was examined.
activity of the enzyme Initially,
and binds equally well 10
to mg of
PPT/PCIMPs 145–155 was added to 8.8 mL of a 1.5 mM BAEE solution (50 mM Tris-HC
the substrate [51,52].
buffer, pH 7.6). The product concentration was monitored using HPLC. The test was con-
3.6. Reusability
ducted consecutively four times. It was observed from the results that the PPT/PCIMPs
Additionally, the reusability of PPT/PCIMPs was examined. Initially, 10 mg of
retained 90% of activity in 540 sec in the first cycle; however, in the subsequent cycles, it
PPT/PCIMPs 145–155 was added to 8.8 mL of a 1.5 mM BAEE solution (50 mM Tris-HCl
slightly dropped.
buffer, pH 7.6). The activityconcentration
The product of the protein wassustained
monitoredafter
usingfour
HPLC.cycles is aswas
The test shown in
Figure 7.
conducted consecutively four times. It was observed from the results that the PPT/PCIMPs
retained 90% of activity in 540 sec in the first cycle; however, in the subsequent cycles,
it slightly dropped. The activity of the protein sustained after four cycles is as shown
in Figure 7.
Nanomaterials 2021, 11, x FOR PEER REVIEW
Nanomaterials 2021, 11, 334 10 of 13

Figure 7. Reusability of PPT/PCIMPs145–155 .

Figure 7. Reusability of PPT/PCIMPs145–155.
3.7. Comparison Studies of the Proposed PPT/PCIMPs with Other Methods
The catalytic hydrolysis
3.7. Comparison Studies of performance
the ProposedofPPT/PCIMPs
the fabricated PPT/PCIMPs was compared
with Other Methods
to previous studies (Table 3). For example, Atacan and colleagues modified the surface of
Fe3 O4The catalyticwith
nanoparticles hydrolysis
gallic acid.performance of the
According to their fabricated
research, Km valuesPPT/PCIMPs
of trypsin andwas com
to previous studies (Table 3). For example, Atacan and colleagues
immobilized trypsin were 5.1 and 7.88 mM, respectively, indicating that the immobilized modified the su
trypsin
Fe has less affinity with
3O4 nanoparticles for thegallic
substrate,
acid.which might beto
According attributed to the lossKof
their research, enzyme of tryp
m values

immobilized trypsin◦ were 5.1 and 7.88 mM, respectively, indicating thatfour
flexibility. Although immobilized trypsin retained 92% of its initial activity after the immo
months of storage at 4 C, there was a dramatic decrease in its activity after being reused
trypsin has less times
eight consecutive affinity[49].for the substrate,
In another which
study, trypsin was might be attributed
immobilized on polymerto the
andloss of e
flexibility. Although
grafted magnetic beads, inimmobilized
which the Km fortrypsin retained
immobilized trypsin92% of itstoinitial
was found be 13.6 activity
mM, aft
months of storage at 4 °C, there was
1.4-fold higher than free trypsin, while V maxa dramatic decrease in its activity after being
value was found to be 3946 U/mg, 1.5-fold
lower than
eight for the freetimes
consecutive trypsin,[49].
indicating that a change
In another study, in the affinitywas
trypsin of the enzyme towards
immobilized on polym
the substrate occurred upon its immobilization [50]. In different work, by Bayramoglu and
grafted magnetic beads, in which the Km for immobilized trypsin was found to
colleagues, polymer grafted magnetic beads were activated with glutaraldehyde for the
mM, 1.4-fold of
immobilization higher
trypsinthan free trypsin,
on affinity while to
ligands attached Vmax
the value was found
beads’ surface. to bethe
Moreover, 3946 U/m
fold lowerand
reusability than for the
activity werefree trypsin,
relatively indicating
good thatwhen
in this study a change
comparedin the affinity
to the above of the e
work. The K and
towards them substrate V values obtained for the immobilized trypsin
max occurred upon its immobilization [50]. In different were of 16.8 mM work, b
and 5115 U/mg, 1.8-fold higher and 1.5-fold lower than free trypsin, respectively. The Km
ramoglu and colleagues, polymer grafted magnetic beads were activated with glut
values could be explained by the fact that there existed conformational changes during
hyde
enzyme forimmobilization
the immobilization
[53]. of trypsin on affinity ligands attached to the beads’ s
Moreover, the reusability and activity were relatively good in this study when com
Tableto Comparison
3. the above studies
work.ofTheproposed PPT/PCIMPs
Km and with other
Vmax values methods.for the immobilized trypsin
obtained
Trypsin/Immobilized Trypsin 16.8 mM Km and 5115 U/mg, V1.8-fold max
higher and 1.5-fold
kcat (s− 1) lower
kcat /K m (mM
than
− 1 s−1 ) free trypsin, respe
Reference
BPT/Immobilized BPT The5.1/7.88
Km values
mM could23/18.3
be explained
mM min−1 by the fact - that there- existed conformational [42] c
BPT/Immobilized BPT during enzyme
9.7/13.6 mM immobilization
5890/3946 U/mg[53]. - 607/290 [43]
BPT/Immobilized BPT Upon a comprehensive evaluation of kinetic parameters, it was[44]
9.3/16.8 mM 7345/5115 U/mg - - evident that
−3 /1.47 × 10−3 mM s−1
PPT and PPT/PCIMPs 233–245+G
gant helical
0.36/0.42 mM cavities
3.2 × 10imprinting strategy 2.6/>1.16
created recognition 7.32/2.79 sites This on study
the MPs sur
which
Abbreviations: Bovine the Trypsin
Pancreas enzymes(BPT),were
Porcine tightly
Pancreatic bound. Moreover,
Trypsin (PPT), it was
Note: U is defined achieved an improved c
as µmol.

hydrolysis in comparison to other previous studies. The best performance of PPT/P

Upon a comprehensive evaluation of kinetic parameters, it was evident that the
for hydrolysis
elegant of BAEE
helical cavities had the
imprinting following
strategy values forsites
created recognition theon kinetic
the MPs parameters
surface, in Km, V
kwhich
cat values were 0.42
the enzymes were mM,
tightly1.4 μM.s
bound. −1 , and 1.16.s
Moreover, −1 . Additionally,
it was achieved an improved PPT/PCIMPs-im
catalytic
materials exhibited stable catalytic activity and reusability.
hydrolysis in comparison to other previous studies. The best performance of PPT/PCIMPs
for hydrolysis of BAEE had the following values for the kinetic parameters Km , Vmax , and
Tablekcat values were 0.42
3. Comparison mM, 1.4
studies µM·s−1 , and
of proposed 1.16·s−1 . Additionally,
PPT/PCIMPs with otherPPT/PCIMPs-imprinted
methods.
materials exhibited stable catalytic activity and reusability.

Trypsin/Immobilized kcat/Km
Km Vmax kcat (s−1) Refe
Trypsin (mM−1 s−1)

BPT/Immobilized BPT 5.1/7.88 mM 23/18.3 mM min−1 - - [4

Nanomaterials 2021, 11, 334 11 of 13

4. Conclusions
In conclusion, a state-of-the-art method for point immobilization of enzymes on
magnetic particles is accomplished. To maintain the catalytically competent state of an
enzyme, an immobilized enzyme at a maximum degree of freedom is the ultimate choice.
Our systems operate by binding enzyme partially and maintaining the remaining part of
the enzyme free. The combination of site fixation with the use of conformation-specific
PCIMPs could boost the catalytic process in many enzymes. Moreover, the experimental
results also indicated the inhibition effect on capturing at the α-helix region to interfere
with catalysis flexibility. The Km of PPT/PCIMPs233–245+G was slightly higher than that of
PPT, resulting in lower diffusion limitations of the substrate and product molecules from
the polymer matrix to forming an enzyme-substrate complex. Consequently, this method
is an appropriate choice for realizing the relationship between each segment’s flexibility
and catalytic activity. We thus believe the PCIMPs strategy can be more widely applied in
green chemistry as a nano biocatalyst.

Author Contributions: Conceptualization, D.-F.T.; methodology, K.R.K., P.-Y.H., Y.-L.C., C.H.W.

and W.L.; software, K.R.K. and P.-Y.H.; validation, K.R.K., P.-Y.H., Y.-L.C., C.H.W. and W.L.; formal
analysis, K.R.K., P.-Y.H., Y.-L.C., C.H.W., W.L. and R.K.K.; resources, C.-H.L. and D.-F.T.; data curation,
K.R.K., C.-H.L., D.-F.T., P.-Y.H., Y.-L.C., C.H.W., W.L. and R.K.K.; writing—original draft preparation,
K.R.K., D.-F.T., R.K.K. and C.-H.L.; writing—review and editing, K.R.K., D.-F.T., R.K.K. and C.-
H.L.; visualization, K.R.K., D.-F.T., R.K.K. and C.-H.L.; supervision, D.-F.T., and C.-H.L.; project
administration, D.-F.T.; funding acquisition, D.-F.T. All authors have read and agreed to the published
version of the manuscript.
Funding: This work is partially supported by the Taiwan Ministry of Science and Technology (MOST
106–2113-M-259–005).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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